Sutures have been used as a conventional surgical means for uniting tissues and surgical margins, as hemostatic aids, and for blocking or ligation. However, sutures suffer from many drawbacks. For example, sutures may be incompatible with the tissue, causing fistula or granuloma, sutures may cut through parenchymal and inflammatory tissues, absorbable suture material may disintegrate prematurely and produce dehiscence of the wound, and closely spaced sutures may cause tissue ischemia resulting in necrosis of the wound margins. Suturing is also time-consuming.
In order to overcome the above-mentioned shortcomings of sutures, various attempts at developing suitable substitutes have been made. One goal has been the development of a tissue glue which ensures union of the tissue without causing any damage thereto.
Cyanoacrylate-based substances have been commonly used as a fibrin glue. However, these substances are toxic to the tissue and cannot be absorbed (J. A. Collins, et al., "Cyanoacrylate Adhesives as Topical Hemostatic Aids", Surgery 65, 260-263, 1969). Thus, this type of tissue glue was found to result in the growth of granulated tissue in response to the foreign substance, rejection of the cyanoacrylate, fistula formation and local suppuration.
As early as 1909, it was realized that "fibrin powder" could be utilized to achieve blood clotting and wound healing (H. Matras, "Fibrin Seal: The State of the Art", J. Oral Maxillofac Surg 43, 605-611, 1985). Others later used fibrin tampons and thin fibrin plaques to control bleeding in parenchymal organs (see, e.g., E. G. Grey, "Fibrin as a Hemostatic in Cerebral Surgery", Surg Gynecol Obstet 21, 452-454, 1915). Another attempt involved the uniting of rabbit nerve with chicken plasma and chicken embryo extract (J. Z. Young, et al., "Fibrin Suture of Peripheral Nerves", Lancet 239, 126-128, 1940). Other work involved autologous and homologous rabbit plasma (I. M. Tarlov, et al., "Plasma Clot and Silk Suture of Nerves", Surg Gynecol Obstet 76, 366-369, 1943). In 1944 the first use was made of a combination of fibrinogen and thrombin for anchoring skin grafts, but the results failed to demonstrate a good adhesive effect (see E. P. Cronkite, et al., "Use of Thrombin and Fibrinogen in Skin Grafting", JAMA 124, 976-980, 1944, and R. T. Tedrick, et al., "Fibrin Fixation of Skin Transplants", Surgery 15, 90-93, 1944).
Due to advances in basic research, it is now possible to prepare highly concentrated plasma products and isolate some coagulation factors. Rabbit cryoprecipitate solution and an equal amount of thrombin solution have been used together with fascicular adaptation to reunite a rabbit nerve stump. This procedure was later applied on a human. In the human application, autologous plasma cryoprecipitate solution was first used, but since the clottable substances were found to be insufficient, homologous cryoprecipitate solution from pooled single-donor plasmas was subsequently used to obtain higher concentration for better tensile strength. Later, fibrin glue or sealant became more widely known.
Fibrin glue or sealant was successfully adapted for use in microvascular surgery. Others later combined suturing and sealing when applying the procedure in neurosurgery for extra-intracranial anastamosis, and on the dura repair, satisfactory results were obtained using fibrin sealant.
Fibrin sealant has three components: fibrinogen concentrate, calcium chloride and thrombin. These components mimic the final common pathway of the clotting cascade, i.e. the conversion of fibrinogen to fibrin (see, e.g., R. W. Colman, et al., Hemostasis & Thrombosis (2d ed.), 1987). In vitro, fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells. Fibrinogen also has been found to stimulate fibroblast growth. The surface protein of fibroblasts has been found to contain fibronection.
Various publications discuss the clinical applications of fibrin sealant, but only a few mention the binding or tensile strength of the fibrin sealant (see, e.g. Jorgensen, et al., "Mechanical Strength in Rat Skin Incisional Wounds Treated with Fibrin Sealant", J. Surg Research 42, 237-241, 1987 and Bense, et al., "Effect of Fibrin Sealant on the Tensile Strength of Rat Visceral Pleura", Scand J. Thor Cardiovasc Surg 21, 179-180, 1987). Heretofore, there has also been a lack of data concerning the required concentration of fibrinogen for tissue binding and the necessary tensile strength at this fibrinogen concentration for use in various tissues.
In preparing fibrin sealant, thrombin of bovine origin is diluted with calcium chloride, with concentrations dependent on the tissue to be applied and the time of clotting. Equal amounts of fibrinogen concentrate and thrombin diluted in calcium chloride are used for clinical application. When the two components are mixed, thrombin converts fibrinogen to fibrin so that clotting is initiated and the mixture solidified. Meanwhile, in the presence of calcium ions, thrombin activates factor XIII to factor XIIIa. Activated factor XIIIa together with thrombin catalyzes the crosslinkage of fibrin and increases the strength of the clot. During would healing the clot material undergoes gradual lysis and is completely absorbed.
A major application of fibrin sealant is in surgery as a hemostasis aid, especially in thoracic-cardiovascular surgery, and in traumatic surgery (liver or spleen injury). In other areas of medicine, fibrin sealant is used as a tool to facilitate hemostasis, permit tissue fixation, enhance implant material growth, stimulate fibroblast growth and as an embolization material. Applications include orthopedic surgery, neural surgery, periodontal surgery, cerebral surgery, sinus or fistula obturation in proctologic and general surgery, chest surgery and genitourinary surgery, skin grafting in burn patients, punch hair grafting in plastic surgery, closure of corneal incisions in eye surgery, repair of lymph leak in general surgery and in myringoplasty in ear surgery.
Although there are great advantages to using fibrin sealant in clinical medicine, it is prohibited to use the commercially available product from pooled human plasma in the United States because of potential transmission of hepatitis B, acquired immunodeficiency syndrome (AIDS), and other transfusion transmittable diseases. The Food and Drug Administration (FDA) regulations have required that all plasma protein fractions shall receive heat treatment for not less than 10 or more than 11 hours at an attained temperature of 60.degree.+/-0.5.degree. C. to inactivate infectious agents.
Commercially available fibrinogen is prepared from the plasma pooling of a large number of donors, which has high potential for disease transmission. In addition, fibrinogen will not tolerate the ten hours of heating to 60.degree. C. used to inactivate the hepatitis virus in other blood fractions. Studies have indicated that this product was a source of hepatitis transmission (7.8% of post-transfusion hepatitis rate). Under these circumstances, the FDA revoked all licenses for the manufacture of human fibrinogen since Jun. 30, 1978.
In Europe, fibrinogen product is commercially available as a fibrinogen concentrate kit ("Tisseel", Immonu AG, Vienna, Austria) prepared from pooled fresh frozen plasma. The tensile strength for Tisseel is 900/g/cm.sup.2. Since this commerical fibrinogen concentrate is not available in the United States because it is currently not licensed by the FDA, alternative methods such as chemical precipitation and cryoprecipitation have been used to prepare fibrinogen concentrate.
Fibrinogen is one of the three main protein constituents of plasma. The major constituent, albumin (ALB), occurs in a concentration of approximately four percent. The plasma globulins are present in a concentration of about 2.5 percent and are particularly associated with the processes of immunity. Fibrinogen occurs in much smaller amounts, with its concentration in human plasma being about 0.4 percent.
Several authors have discussed fibrinogen/fibrinogen interaction and fibrinogen interaction with other proteins. Aggregation of fibrinogen at pH 5.7 and low ionic strength (&lt;0.3) has been found. A disulfide bond between fibrinogen molecules in cold-insoluble fibrinogen fraction has been demonstrated. It has been thought that the cold-insoluble precipitate that formed from normal plasma was a reaction between cold-insoluble globulin (CIg), fibrinogen and fibrin.
The plasma proteins can be separately isolated by: 1) organic solvents such as methanol or ethanol at low temperature using Cohn's fractionation, 2) cryoprecipitation, 3) chemical precipitation of plasma with salts such as ammonium sulfate, potassium phosphate, and sodium citrate, and 4) other methods. The solubility of the plasma proteins in these substances decreases in the order of albumin, globulin, and fibrinogen. The latter precipitates first and albumin last upon the addition of increasing amount of the precipitating agent.
1. Ethanol Fractionation (Cohn's fractionation)
In this process, 1,000 to 1,500 liters of 4,000-6,000 human source plasma are pooled and treated sequentially in the cold with various concentrations of ethanol and buffers to precipitate fractions containing different plasma proteins. Fibrinogen is the first material precipitated and harvested at-5.degree. C. with 25% ethanol at a pH of 6.9. Variables determining the precipitation of proteins are ethanol concentration, pH, temperature, ionic strength and protein concentration.
2. Cryoprecipitation
The standard cryoprecipitation method has been primarily used to prepare antihemophilic factor (Factor VIII). Cryoprecipitate also has been known as a source of fibrinogen. The cryoprecipitate method can be also used to prepare fibrinogen concentrate. It is known that some factors might affect the yield of Factor VIII, such as ABO blood grouping, freezing and thawing conditions (see Kasper, et al., "Determinants of Factor VIII Recovery in Cryoprecipitate", Transfusion 15, 312-322, 1975, and Rock, et al., "Variations in Cryoprecipitate Production", Transfusion 17, 50-53, 1977). With respect to Factor VIII preparation, others have studied freezing and thawing conditions (see Brown, et al., "Antihaemophilic Globulin: Preparation by an Improved Cryoprecipitation Method and Clinical Use", Br Med J 2, 79-85, 1967). However, all the factors for cryoprecipitation are not known.
It has been observed that when frozen plasma is thawed in the cold at 4.degree. C., most of the Factor VIII remains in the cold-insoluble precipitate. This precipitate also contains variable amounts of fibrinogen ranging from 100 to 300 mg/single donor unit of cryoprecipitate. It has become routine to prepare anti-hemophilic factor (Factor VIII) and fibrinogen using the cryoprecipitation method in the blood bank using a closed system of plastic bags to maintain the sterility of the product from collection of the whole blood from the donor. See, e.g.: Rousou, et al., "Fibrin Glue: An Effective Hemostatic Agent for Nonsuturable Intraoperative Bleeding", Ann Thorac Surg. 38, 409-410, 1984; Lupinetti, et al., "Cryoprecipitate-Topical Thrombin Glue", J. Thorac Cardiovasc Surg 90, 502-505, 1985; Ness, et al., "Cryoprecipitate as a Reliable Source of Fibrinogen Replacement", JAMA 241, 1690-1691, 1979; Brown, et al., "Antihaemophilic Globulin: Preparation by an Improved Cryoprecipitation Method and Clinical Use", Br Med J 2, 79-85, 1967; Ness, et al., "Fibrinogen in Cryoprecipitate and Its Relationship to Factor VIII (AHF) Levels", Transfusion 20, 93-96, 1980; Carlebjork, et al., "Freezing of Plasma and Recovery of Factor VIII", Transfusion 26, 159-162; Masure, "Human Factor VIII Prepared by Cryoprecipitation", Vox Sang 16, 1-9, 1969, and; Williams, et al., "A New and Improved Method for the Preparation of Autologous Fibrin Glue and Further Applications.", Exhibit Presentation, 71st Annual Clinical Congress of the American College of Surgeons, 1985.
3. Chemical Precipitation
Human fibrinogen can be precipitated from human plasma by ammonium sulfate, polyethylene glycol, plyvinyl-pyrrolidone, and barium/magnesium sulfate. Entering the closed blood bag system for the addition of chemicals opens the system to the potential for bacterial contamination. Small amounts of fibrinogen concentrate solution (0.5-1.9 ml) can be prepared using these methods, but the side effects and safety due to the chemical substances as well as bacterial contamination opportunities are of great concern.
4. Other Methods
Sporadic reports have mentioned the use of the following methods to prepare purified fibrinogen: chromatography, polyelectrolyte fraction technology, recombinant DNA technology and ion exchange chromatography. See C. Th. Smit Sibinga, et al., "Plasma Fractionation and Blood Transfusion", Martinus Nijhoff Publishers, Northland, 1985.
As mentioned above, several methods have been developed for the isolation of purified fibrinogen. However, these have numerous drawbacks that make them inapplicable in clinical use, such as disease transmission (heat treatment intolerable), bacterial contamination (using open system), chemical toxicity and safety, inadequate product volume, time consumption, and cost. Disease transmission is one of the main concerns and the reason the FDA has not approved the commercially prepared fibrinogen concentrate (Tisseel) for use in this country.
Among the methods described previously, the cryoprecipitation method is the simplest and most economic way to make concentrated fibrinogen. Most U.S. blood banks use cryoprecipitate as the fibrinogen (FBG) source for fibrin glue which contains less FBG (260-2,500 mg/dl) compared to Tisseel (7,000-10,000 mg/dl).
Fibrinogen concentrate can be prepared from random single-donor fresh frozen plasma or autologous plasma in sufficient quantity to meet some surgical demand. According to the Standards of the American Association of Blood Banks, fibrinogen concentrate can be currently stored for up to 5 years at -80.degree. C. or at least 5 days at 4.degree. C. until it is needed. Cryoprecipitate contains Factor VIII and fibrinogen and is used to supply fibrinogen in patients with hypofibrinogemia and also as an alternative source of fibrinogen concentrate for fibrin sealant in the United States.
However, traditional cryoprecipitation suffers from problems including the recovery of only small amounts of fibrinogen having low tensile strength when using single-donor cryoprecipitate to prepare fibrin sealant. Further, the fibrinogen concentrates prepared by traditional cryoprecipitation have a concentration range of 260-2,500 mg/dl. This is not an adequate concentration for applying this product as a tissue sealant over highly vascular areas. High fibrinolytic activity over that area breaks down the fibrin clot very quickly. These concentrates have a tensile strength of around 120 gm/cm.sup.2 which is usually not sufficient for surgical applications.
The following patents reflect the state of the art of which applicant is aware insofar as these patents appear germane to the patent process. However, it is respectfully stipulated that none of these patents teach singly nor render obvious when considered in any conceivable combination the nexus of the instant invention as set forth hereinafter.
______________________________________ INVENTOR U.S. PAT. NO. ISSUE DATE ______________________________________ Anderson, et al. 3,920,625 1975 Garber, et al. 4,025,618 1977 Seufert 4,141,887 1979 Shanbrom 4,188,318 1980 Rose, et al. 4,627,879 1986 ______________________________________
None of the prior art resolves the longstanding and vexing problem that comes from the inefficient extraction of fibrinogen. Optimization of fibrin or fibrinogen extraction particularly as outlined hereinafter, allows for the autologous provision of fibrin from an individual immediately prior to surgery such that the fibrin is extracted from the patient and the residual blood components are restored to the individual with no discernable adverse effects that would mitigate against a commencement of the operation.